Chromophoric dye molecules are being tested as resonance Raman "probes" of biomacromolecules. The chromophoric dyes are bound to the biomolecule under investigation and the resonance Raman spectrum of the chromophoric dye in the complex is determined. We have used this approach to identify bonding interactions between the probe and the macromolecule. Sites of covalent attachment between chromophores and carbonic anhydrase and carboxy peptidase have been determined. The method has also been used to determine the ionization state of the bound chromophore in both reversible and irreversible enzyme-probe complexes of carbonic anhydrase and carboxypeptidase. Stereochemical assignment has also been made using this approach. We have determined the stereochemistry of chromophoric substrate bound to glyceraldehyde phosphate dehydrogenase and chymotrypsin, allowing investigation of the deformation of a small molecule upon binding to the enzyme. In addition, metal-ligand interactions have been determined in studies of complexes of probe molecules with liver alcohol dehydrogenase.